Type-l cyclic GMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) signaling in vascular smooth muscle cells (VSMC). Two isoforms, PKG-la and PKG-I[unreadable], are believed to mediate the NO-dependent smooth muscle relaxation, inhibition of cell growth, and increase in contractile protein gene expression. However, inflammatory cytokines such as interleukin-1 (IL-1) are known to stimulate VSMC growth and block contractile protein gene expression. Our laboratory has shown that inflammatory cytokines also inhibit PKG-I expression in VSMC suggesting that at least one mechanism of action of these cytokines is to suppress PKG-I and induce a fibroproliferative VSMC phenotype. The mechanisms by which cytokines inhibit VSMC expression of PKG-I will be explored in this proposal. This is a basic and fundamental science proposal to identify in VSMC the molecular mechanisms regulating PKG-I expression in these cells. The central hypothesis is that IL-1 and other cytokines down-regulate PKG-I expression through two major mechanisms: first, through increased production of cGMP in the cells (via and induction of type II NO synthase or iNOS), PKG-I is ubiquitinylated and degraded in the 26S proteasome, and second, through suppression of AU-rich binding proteins that bind to and stabilize the PKG-I mRNA. Aim 1 will examine the mechanisms by which PKG-I is targeted for ubiquitinylation. Our hypothesis is that the binding of cGMP, specifically to the PKG-la isoform, induces autophosphorylation of the regulatory domain of the kinase which targets it for ubiquitinylation. We will identify the autophosphorylation site, the ubiquitinylation site, and the E3 ligase that binds autophosphorylated PKG-la. The effects of down-regulation of PKG-la on VSMC phenotypic properties will be assessed. In Aim 2, we will examine the role of the 3'-untranslated region (UTR) of the PKG-I mRNA in stabilizing both the message and the protein. Our hypothesis is that specific AU-rich binding proteins stabilize PKG-I mRNA and that inflammatory cytokines down-regulate these stabilizing proteins thus producing a destabilized mRNA and suppressed protein expression. In both of these mechanisms, PKG-la and I[unreadable] may be down-regulated in response to inflammation and increased cytokine production. These effects may facilitate the modulation of contractile VSMC to synthetic VSMC. [unreadable] [unreadable] [unreadable]